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Cite This: J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
Mechanistic Exploitation of a Self-Repairing, Blocked Proton Transfer Pathway in an O2‑Tolerant [NiFe]-Hydrogenase Rhiannon M. Evans,† Philip A. Ash,† Stephen E. Beaton,† Emily J. Brooke,† Kylie A. Vincent,† Stephen B. Carr,*,‡,§ and Fraser A. Armstrong*,† †
Department of Chemistry, University of Oxford, Oxford OX1 3QR, United Kingdom Research Complex at Harwell, Rutherford Appleton Laboratory, Harwell, Didcot OX11 0QX, United Kingdom § Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom Downloaded via UNIV OF READING on August 2, 2018 at 19:29:41 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
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ABSTRACT: Catalytic long-range proton transfer in [NiFe]hydrogenases has long been associated with a highly conserved glutamate (E) situated within 4 Å of the active site. Substituting for glutamine (Q) in the O2-tolerant [NiFe]hydrogenase-1 from Escherichia coli produces a variant (E28Q) with unique properties that have been investigated using protein film electrochemistry, protein film infrared electrochemistry, and X-ray crystallography. At pH 7 and moderate potential, E28Q displays approximately 1% of the activity of the native enzyme, high enough to allow detailed infrared measurements under steady-state conditions. Atomiclevel crystal structures reveal partial displacement of the amide side chain by a hydroxide ion, the occupancy of which increases with pH or under oxidizing conditions supporting formation of the superoxidized state of the unusual proximal [4Fe−3S] cluster located nearby. Under these special conditions, the essential exit pathway for at least one of the H+ ions produced by H2 oxidation, and assumed to be blocked in the E28Q variant, is partially repaired. During steady-state H2 oxidation at neutral pH (i.e., when the barrier to H+ exit via Q28 is almost totally closed), the catalytic cycle is dominated by the reduced states “Nia-R” and “Nia-C”, even under highly oxidizing conditions. Hence, E28 is not involved in the initial activation/deprotonation of H2, but facilitates H+ exit later in the catalytic cycle to regenerate the initial oxidized active state, assumed to be Nia-SI. Accordingly, the oxidized inactive resting state, “Ni-B”, is not produced by E28Q in the presence of H2 at high potential because Nia-SI (the precursor for Ni-B) cannot accumulate. The results have important implications for understanding the catalytic mechanism of [NiFe]-hydrogenases and the control of longrange proton-coupled electron transfer in hydrogenases and other enzymes. shorter than for an electron;9 hence a proton pathway requires closely spaced donor−acceptor groups (mobile side chains and water molecules) whereas electron-transfer sites are typically 10−14 Å apart.10 Neutral gas molecules, whether substrates or inhibitors, are expected to enter and leave the active site via preferred hydrophobic tunnels.11−13 Additionally, and especially for the special ‘O2-tolerant’ class of [NiFe]-hydrogenases that can function continuously in the presence of O2,14 (and thus act as hydrogen oxidases15,16) hydrophilic pathways must exist to ensure rapid escape of water molecules following O2 attack.12,17,18 The Escherichia coli membrane-bound [NiFe]-hydrogenase (MBH) “Hyd-1” is O2-tolerant with a strong bias toward hydrogen oxidation at neutral pH, whereas the O2-sensitive MBH “Hyd-2” from E. coli operates in both directions.19,20 The active site, housed in the large subunit of the
1. INTRODUCTION The cycling of hydrogen (H2) by microorganisms has strong connections to biotechnology, energy and health.1−3 The metalloenzymes known as hydrogenases offer a paradigm for fast, efficient and reversible4 hydrogen electrocatalysis requiring only a minimal overpotential in either direction, and set a very high standard for designing catalysts based on abundant, nonplatinum metals.5−7 Molecular H2 activation is the simplest of all proton-coupled electron-transfer reactions. Hydrogenases operate in neutral water and use a heterolytic mechanism (H2 ⇌ H+ + H− ⇌ 2H+ + 2e−)8 so it is highly significant that their active sites are deeply buried and almost completely sealed from solvent. In fact, they are remarkable among all enzymes in that H2, the smallest of molecules, is solely formed from (or converted into) four quantum particles that must hop and tunnel through the protein. The specific routes taken by protons and electrons between solvent and active site reflect the fact that the characteristic tunneling distance for a proton is about 45 times © XXXX American Chemical Society
Received: May 10, 2018
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DOI: 10.1021/jacs.8b04798 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
Article
Journal of the American Chemical Society
Figure 1. Schematic representation of the orientation of E28 in native Hyd-1 (A), in which the noncoordinating residues E28 and R509 so far established to be essential for catalysis are included. Distances (gray) between the carbonyl-O atoms of E28 and the nearest Fe atom of the [4Fe− 3S]-6Cys proximal Fe−S cluster and the cysteine-S of Ni-coordinated cysteine 576 are shown (pdb.5A4M). The atom which bridges the Ni and Fe is denoted as ‘X’ to represent the varied identification of the ligand depending on the oxidation state (as shown in the catalytic cycle, B) and presence of various inhibitory small molecules. (B) In the catalytic cycle, the catalytically active (a) states are denoted “Nia” whereas the inactive oxidized states generated anaerobically at high potential (Ni-B) or upon the binding of oxygen (Ni-B or Ni-A depending on the number of electrons immediately available to reduce the attacking O2, see refs65,69) are shown in the gray box. Each catalytic state is shown in a black box: the lower half represents the current consensus on the minimal [Ni, Fe, H] unit of the active site, including oxidation states and bonding, the upper half represents the proton acceptor(s) not associated with the minimal unit. During H2 oxidation, H2 binds to the Nia-SI state (1), heterolytic activation of H2 requires deprotonation by a nearby base (‘:B1’, the identity of which is still under debate17,23,27,70,71) forming Nia-R which has a bridging hydride (2).71 The subsequent stage of the catalytic cycle in which the proton on B1 leaves the active site is unknown at present (so we denote the protonated base in parentheses ([H+]:B1)), though B1 must be deprotonated prior to stage 2 in order for the next molecule of H2 to be activated. Following electron transfer Nia-C is formed (3). The bridging hydride of Nia-C migrates as a proton, the initial proton acceptor proposed to be a terminal cysteine thiolate (4),27,70 and the resulting Nia-L state is proposed to contain a dative metal−metal bond.72 Finally, a second electron is transferred to the proximal Fe−S cluster to form Nia-SI (5).
active site to the biological membrane or redox partner protein. The Fe−S chain comprises three Fe−S clusters termed “proximal”, “medial” and “distal” according to their distance from the active site. In contrast to this clearly marked pathway for electrons, our insight into long-range proton transfer owes much to the seminal work of Dementin and co-workers24 who showed that a highly conserved glutamate23 (E28 in E. coli Hyd-1, Figure 1A) located at the interface of the large and small subunits, and in direct contact with an extensive network of water molecules (see later and Figure S1) is critical for proton transport from the active site during H2 oxidation. Mutation of E28 to glutamine (carboxylate-to-amide) in the [NiFe]-hydrogenase from Desulfovibrio fructosovorans resulted in a protein that could cleave H2 within the active site, but was unable to exchange protons with solvent, resulting in >99.9% lowering of the H2 oxidation rate. No electrochemical or structural data were presented. There has since been compelling support for a key role for this residue in proton
heterodimeric protein complex, has similar architecture in all cases, the essential features being summarized in Figure 1A.17,21,22 The Ni and Fe atoms are anchored to the protein by four cysteine thiolates, two of which are bridging ligands while the other two are terminal to the Ni: the Fe atom is further ligated by one CO and two CN− ligands. Depending on the stage of the minimal catalytic cycle shown in Figure 1B, a fifth ligand to the Ni may be present, in a bridging position between the Ni and Fe atoms (the Fe remains low-spin Fe II throughout). Immediately above the metal atoms lies the outer coordination shell “canopy” that includes the guanidinium group of arginine 509, recently demonstrated to be essential for fast H2 oxidation by Hyd-1.17,23 Whereas the detailed mechanism by which the H−H bond is formed or cleaved remains to be resolved, much more is known about the pathways of electrons to and from the buried active site. In MBHs, electrons are transferred via a chain of Fe−S clusters housed in the small subunit that connects the B
DOI: 10.1021/jacs.8b04798 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
Article
Journal of the American Chemical Society
recently reported.21 The resulting native Hyd-2, previously termed “Hyd-2-NOP”, (native, “overproduced”), is simply denoted “Hyd-2”. For the Hyd-2 variant E14Q, strain HJ001-hyp hybC was mutated as outlined in Table S1. Enzymes Hyd-2 and E14Q were purified by Niaffinity chromatography, and for subsequent structural studies underwent additional size-exclusion chromatography.21 2.2. Steady State Solution Assays. Turnover rates for H2 oxidation were measured by anaerobic steady-state solution assays in an N2-filled glovebox (Belle Technologies, O2
